Autonomously-activating bioluminescent reporters to enable continuous, real-time, non- invasive brain cell imaging Project Summary This Small Business Innovation Research (SBIR) Phase I project proposes to develop a set of genetically encoded, self-exciting bioluminescent (autobioluminescent) optical imaging reporters that will enable continuous neuron and astrocyte specific imaging without perturbing endogenous cellular metabolism, while using common laboratory equipment. The treatment and management of brain disorders imposes enormous financial and social costs. The NIH therefore launched the BRAIN initiative to develop a new generation of innovative research tools and therapies that enable brain-cell-specific imaging, spatiotemporal tracking, and continuous, non-invasive cell monitoring to facilitate new methods for evaluating brain cell physiology and new means to identify relative connectivity. The current generation of brain cell imaging tools, which rely on fluorescent and luciferin-luciferase bioluminescent chemistries, are incapable of achieving the NIH BRAIN initiative?s goals. Fluorescent imaging modalities require an excitation light to trigger their emission signal. This excitation is difficult to deliver to the brain and causes high levels of background autofluorescence that restricts signal discrimination. Bioluminescent imaging, which has a greater signal-to-noise ratio due to a lack of background bioluminescence in tissue, is similarly limited in that it can only produce an emission signal in the presence of an externally supplied substrate (luciferin). Repeated application of this substrate results in discontinuous signaling and substrate distribution kinetics that are challenging to replicate. Therefore, to meet the needs of the NIH BRAIN initiative and overcome the limitations restricting existing brain cell imaging tools, 490 BioTech will reengineer our synthetic autobioluminescent genetic operon to create a new type of reporting technology that requires no external stimulation (e.g., light or luciferin substrate) to provide continuous, non-invasive, brain-cell-specific optical monitoring. The genetic topology of the synthetic autobioluminescent operon will be systematically optimized to maximize signal output and minimize its effects on the host brain cell?s metabolism. Using this technology, neuron and astrocyte specific autobioluminescent differentiation reporter platforms will be developed that will enable researchers to track the onset of brain cell differentiation and the subsequent fate of descendant cells. These autobioluminescent brain-cell-specific technologies will be validated using 3D cell culture to demonstrate their utility and generate proof-of-principle data. At the conclusion of this project we will deliver a set of genetic constructs endowing brain-cell-specific, continuously autobioluminescent phenotypes that researchers can utilize to continuously and non-invasively label and track cellular location, physiology, and connectivity.